Comprehensive analysis of small molecule metabolites in complex biological systems can provide important insight that may otherwise be difficult or impossible using targeted analysis of specific compounds. Conventional analysis of metabolites using Gas Chromatography and Mass Spectrometry (GC/MS) involves differentiating small molecules having labels, or tags, whereby the label lowers the vapor pressure of small target molecules to enhance transmission through the analytical system. Increasingly, Liquid Chromatography/Mass Spectrometry (LC/MS) is being used for metabolic analysis due in part to widespread availability and compatibility with biological samples. Currently, however, a need exists for comprehensive relative quantification of metabolites using LC/MS analysis techniques.
Comprehensive analyses of small molecule metabolites in complex systems may also provide important insight into biological systems. The desired outcomes of such metabolomics studies are the detection, identification, and precise quantification of a large number of metabolites with diverse chemical structures and broad concentration ranges. Much work has focused on identifying as many metabolites as possible and many NMR and mass spectrometric techniques have been employed. (Birkemeyer C et al., Trends Biotechnol. 2005, 23, 28-33; Rochfort S J, Nat. Prod. 2005, 68, 1813-1820; Sumner L W et al., Phytochemistry 2003, 62, 817-836; and Weckwerth W et al., Curr. Opin. Biotechnol. 2002, 13, 156-160).
Mass spectrometry provides excellent sensitivity and good identification capabilities, but special considerations are necessary to deal with the complexity of metabolomic samples and with quantification. The coupling of a separation technique to mass spectrometry is an important common method of dealing with the sample complexity issue. Direct analysis of metabolic extracts by mass spectrometry has been reported. (Aharoni A et al., Omics 2002, 6, 217-234; and Vaidyanathan S et al., Anal. Chem. 2001, 73, 4134-4144). GC-MS has been used extensively for profiling non-polar compounds as well as some polar ones (i.e., after derivatization) in order to increase volatility. (Fiehn O et al., Nature Biotechnol. 2000, 18, 1157-1161; and Koek M M et al., Anal. Chem. 2006, 78, 1272-1281).
Increasingly, LC-MS is being used for metabolomic analyses owing to its compatibility with a wide-range of analytes in biological samples. (Dalluge L L et al., Chromatogr. A 2004, 1043, 3-7; Lafaye A et al., Anal. Chem. 2005, 77, 2026-2033; Tolstikov V V et al., Anal. Biochem. 2002, 301, 298-307; von Roepenack-Lahaye E et al., Plant Physiol. 2004, 134, 548-559; Wang W X et al., Anal. Chem. 2003, 75, 4818-4826; Want E J et al., Anal. Chem. 2006, 78, 743-752; and, Wu L et al., Anal. Biochem. 2005, 336, 164-171).
Another important consideration in mass spectrometric analysis is quantification. Various strategies have been employed for quantitative LC-MS of metabolomic samples. Absolute quantification of an analyte relies upon addition of an internal standard differing only in its isotopic form. That method has been employed in many studies that target a particular compound or a small set of metabolites. (Kita Y et al., Anal. Biochem. 2005, 342, 134-143; and, Rabaglia M E et al., Am. J. Physi. Endocrinol. Metab. 2005, 289, E218-E224).
However, it is impractical to add an isotopic standard for every compound when performing more comprehensive metabolic profiling. Relative quantification between samples is more amenable to analyzing broad classes of compounds, and it often provides very useful biological information. Some researchers have turned to in vivo labeling of metabolites with 2H, 13C, or 15N, and comparing the labeled sample with a control sample that has natural isotopic abundances. (Birkemeyer C et al., Trends Biotechnol. 2005, 23, 28-33; Lafaye A et al., Anal. Chem. 2005, 77, 2026-2033; Wu L et al., Anal. Biochem. 2005, 336, 164-171; and, Mashego M R et al., Biotechnol. Bioeng. 2004, 85, 620-628). That strategy works fairly well for certain organisms such as yeast, bacteria, and some plants, but uniform incorporation of these isotopes into all metabolites for animals is quite difficult/expensive or impossible (e.g., metabolites from humans).
Consequently, some quantitative metabolic profiling has been performed without an isotopic standard. (von Roepenack-Lahaye E et al., Plant Physiol. 2004, 134, 548-559; Wang W X et al., Anal. Chem. 2003, 75, 4818-4826; and, Want E J et al., Anal. Chem. 2006, 78, 743-752). Quantification in this manner is less precise, but adequate reproducibility has been obtained in many cases despite the well-known problem of ion-suppression during electrospray ionization. (Choi B K et al., Chromatogr. A 2001, 907, 337-342; Constantopoulos T L et al., Am. Soc. Mass Spectrom. 1999, 10, 625-634; Sterner J L et al., Mass Spectrom. 2000, 35, 385-391; and, Tang L et al., Anal. Chem. 1993, 65, 3654-3668).
Another strategy for relative quantification is chemical labeling, which has proven to be useful for quantification in genomics (e.g., two color fluorescent dye labeling)(Lockhart D J et al., Nature Biotechnol. 1996, 14, 1675-1680; and, Schena M et al., Science 1995, 270, 467-470) and proteomics (e.g., isotope-coded affinity tags)(Aebersold R et al., Nature 2003, 422, 198-207; Gygi S P et al., Nature Biotechnol. 1999, 17, 994-999). Relative quantification by labeling has seen limited use for metabolomics due in part to the lack of a single functional group present in all metabolites to act as the target for the labeling chemistry.
Isotopic labeling reagents have been employed for relative quantification of peptides and proteins. Cys residues of a protein have been reduced and then alkylated with an isotope (heavy or light) containing affinity tag. Tagged whole proteins were then digested with trypsin, which produces cleavage products c-terminal to Lys and Arg resulting in peptide products of 10-15 amino acids in length. Product peptides originally containing one or more Cys residues would have been tagged whereas those without a Cys residue would not. Light and heavy tagged samples were mixed, and an affinity capture procedure extracted labeled product peptides, which were subsequently analyzed by LC-MS. The ratio between heavy- and light-peptide products yields the relative amounts of each constituent. The peptide sequence is determined in the same MS experiment by fragmentation.
One drawback of this approach is that only peptides containing a Cys residue are labeled. Another drawback of this approach is that the labeling reagent can introduce a chromatographic shift between the light- and heavy-labeled products, which hinders quantification. Another drawback of this approach is that the tag does not increase the charge state of the labeled product in ESI-MS. Another drawback of this approach is that it does not block carboxylic acid functional groups.
One drawback of known labeling reagents is that many do not produce ionizable end-products. For example, known target molecules having ionizable functional groups thereon are converted to non-ionizable functional groups upon reaction with the labeling reagent. Another drawback of known labeling reagents is the need for reaction clean-up. Other known labeling reagents are disadvantaged due to the large size/mass of the tag residue relative to the size/mass of the biological target material, which adversely impacts chromatographic separation. Insufficient chromatographic separation is problematic because labeled target materials would be difficult or impossible to be adequately quantified by mass spectrometry. Another drawback of known labeling reagents is an inability to react with carboxylic acids. Still other labeling reagents locate deuterium atoms on a hydrophobic residue, which can interfere or prevent heavy- and light-labeled end-products from co-eluting. A need also exists for reagents that are compatible with labeling of multiple different functional groups.
The instant invention overcomes these and other disadvantages by providing ionizable, isotopic labeling reagents and labeling reaction products for relative quantification using mass spectrometry.